The growth of various cell lines in the laboratory is critical to pharmaceutical development, clinical practice and research investigation. The production of vaccines against various infections diseases requires the growth in liquid cultures of mass quantities of virus-producing cells. In the clinic, continuous maintenance of cells in culture is imperative for many purposes, such as clinical assessment of immune functions and tissue typing in matching donor tissue to transplantation patients. In the research laboratory, tissue culture media of various formulations are required for experimentation in neurophysiology, bacteriology, microbiology, physiology, immunology, endocrinology, biochemistry and cell biology.
The therapeutic use of various monoclonal antibodies is now beginning. To date, the vast majority of monoclonal antibody producing cell lines have been of murine origin. Although murine monoclonal antibodies can be grown to large numbers in the peritoneal cavity of normal mice, it can be safely predicted that they will be of limited therapeutic efficiency in humans who will recognize the murine antibody as a foreign protein and react against it. Similar reactions in the past have led to serum sickness and anaphylaxis. As a result, efforts are being made to develop human hybridoma monoclonal antibody-producing cell lines. Known human antibody-producing cell lines cannot be grown in mice because the animals' immune response destroys them. They could be grown in nude mice, which, since lacking a thymus, are incapable of reacting against human cell line antigens. However, the high cost of nude mice precludes their exploitation in the development of therapeutic human hybridoma cell line products. As a result, practical production of human monoclonal antibodies will require the in vitro culturing of antibody-producing cell lines, with large-scale cultures being needed for production of therapeutic quantities of the antibodies.
In the past, many of the culture requirements for different mammalian cells and tissues have been identified. For instance, it is now known that cells require various sugars, organic and inorganic salts, metal ions, amino acids and derivatives, vitamins and coenzymes, and carbohydrates. This knowledge has led to the development of various media, such as Roswell Park Memorial Institute 1640 media (hereinafter "1640"), Modified Eagles Medium (hereinafter "MEM") and Dulbecco's Modified Eagle's Medium (hereinafter "DMEM"). In addition to the chemically defined substances in these media, the growth of cells in culture has required the addition of some natural product, such as fetal calf serum (hereinafter "FCS"), to approximate a natural environment for the cell or tissue culture. The FCS is routinely used in many animal cell cultures at a 2 to 35 percent by volume concentration.
Although FCS in 1640, MEM or DMEM supports the in vitro growth of many mammalian cell types, the precise components in the serum that promote cell growth remain undefined. It is known, however, that the serum component of media creates numerous problems in the cell cultures. For example, due to variable conditions in slaughterhouses and herd health, FCS and other sera often contain undiscernible viruses which often produce disease. The viral contamination of FCS is then carried through to the final vaccine or hormone product generated by the cells grown in FCS with obvious calamitous results. For researches interested in strict control of additives to cultured cells, serum represents a source of unknown proteins, polypeptides, hormones, salts, et cetera, thus making strict determination of cell growth requirements impossible. In addition, FCS is often difficult to obtain and is expensive when available. For instance, when used at a 10 percent by volume concentration, its cost can be approximately ten times that of the collective costs of the other compounds and chemicals used in standard tissue culture media.
As a consequence of the drawbacks of the use of FCS in cell and tissue culturing, attempts have been made to develop substitutes for FCS. In one type of culture medium disclosed by Iscove, Guilbert and Weyman in 126 Experimental Cell Research 121 (1980) (hereinafter "Iscove's Medium") fetal calf serum was replaced with bovine serum albumin (hereinafter "BSA") human transferrin, and a mixture of phosphatidylcholines, linoleic acid and cholesterol. A severe limitation of Iscove's Medium is that it is capable of growing only a highly restricted set of cells.
Another serum-free cell culture medium is disclosed by Cartaya in U.S. Pat. Nos. 4,205,126 and Re. 30,985, wherein the medium is composed of amino acids, biotin-folic acid, thyroxine, insulin, hydrocortisone, essential fatty acids, vitamins, and surfactants.
A further serum-free medium is disclosed by Torney et al. in U.S. Pat. No. 3,887,930, which incorporates a water-soluble lipid and an ion-exchange resin. Although the ion-exchange resin is said to be beneficial for culturing monolayer cell lines, the removal of cells from the resin can be very difficult.
Thus, it is a principal object of the present invention to provide a defined, serum-free medium capable of culturing a wide range of cells both in suspension and as cell monolayers.
It is a further object of the present invention to provide a serum-free culture medium capable of growing myeloma cells to be fused to normal cells to create antibodies secreting hybridomas, of growing the resultant hybridoma cells, and of cloning the hybridoma cells.
In addition, it is an object of the present invention to provide a culture medium useful for a wide range of mammalian cell culture experimentation.